Inherited mutations in the Breast Cancer 1 and 2 genes (BRCA1 and BRCA2) confer susceptibility to breast and ovarian cancer, as well as to pancreatic and prostate cancers. The BRCA proteins are required for DNA repair by homologous recombination and this function is intimately connected to tumor suppression. BRCA mutation status is also an important predictor of response to chemotherapy. Inhibition of specific DNA repair pathways has emerged as an elegant and effective strategy to induce synthetic lethality in BRCA mutant cancers. Understanding the molecular basis of BRCA dependent DNA repair is therefore a central issue to cancer etiology and therapy. We have discovered that BRCA1 is targeted to lysine63-linked ubiquitin regions aligning DNA double-strand break chromatin by the RAP80-BRCC36 complex. Deficiency of this complex results in reduced BRCA1 DNA damage localization and genomic instability. Moreover, mutations within several genes that encode constituents of this complex are associated with familial breast cancer. This indicates that ubiquitin recognition at double-strand break chromatin is a bona fide component of BRCA dependent tumor suppression. This proposal will take in vivo, cellular, and structure guided biochemical approaches to understand how DNA double-strand break ubiquitin recognition by the RAP80-BRCC36 complex relates to BRCA dependent homologous recombination repair mechanisms and response to therapy. The studies are intended to elucidate basic DNA repair mechanisms and gain insights into cancer etiology and therapy.